Esta pesquisa é patrocinada pelo Escritório de Pesquisa Biológica e Ambiental, Departamento de Energia dos EUA e por subvenções do Conselho de Pesquisa em Saúde da Virginia Commonwealth. Oak Ridge National Laboratory é gerenciado por UT-Battelle, LLC, para o Departamento de Energia dos EUA sob o n º do contrato DE-AC05-00OR22725.

1. Preparação Mica: <ol><li> Corte a mica (Ciências Microscopia Eletrônica) com uma tesoura para o tamanho necessário para caber o microscópio AFM (cerca de 22 × 30 mm). </li><li> Cleave a mica em ambos os lados, geralmente usando a fita para remover a camada externa, até que apenas suaves camadas ininterrupta permanecem. </li></ol> 2. Preparação da solução de gelatina: <ol><li> Adicione 100 ml de água destilada para um frasco de laboratório. </li><li> Aqueça a garrafa em um forno de microondas até que a água começa a ferver. (Se um microondas não está disponível, a água pode ser aquecido em um copo sobre uma chapa quente.) </li><li> Pesar 0,5 gramas de gelatina (Sigma G-6144, G-2625-2500 ou G) e adicione à água quente destilada. </li><li> Agite suavemente o frasco até que a gelatina esteja completamente dissolvido. </li><li> Arrefecer a solução de gelatina a 60-70 ° C. </li><li> Despeje aproximadamente 15 mL para um copo pequeno (20 mL) em que a mica clivada corte podem ser mergulhados. </li></ol> 3. Mica revestimento: <ol><li> Submergir as praças mica na solução aquecida gelatina e retirar rapidamente de modo que a mica é revestido (Fig. 1a). </li><li> Após o revestimento, o apoio da mica imediatamente, no limite, sobre um papel toalha para secar no ar ambiente (Figura 1b). A mica revestidas de gelatina pode ser usada por pelo menos duas semanas. O excesso de solução de gelatina pode ser mantido refrigerado e usado por aproximadamente um mês, basta aquecer a solução de ações para entre 60-70 ° C. Cuidados devem ser tomados para assegurar que a gelatina não é fervida durante o aquecimento. </li></ol> 4. Montagem de bactérias em mica revestidas de gelatina: <ol><li> Pellet 1 mL de uma cultura bacteriana (0,5-1,0 OD a 600 nm) em 800 a 4500 rcf. Dependendo da bactéria, use o mínimo rcf que irá agregar as células em ~ 5 minutos. </li><li> Lavar o sedimento em um mesmo volume de água filtrada ou deionizada buffer. </li><li> Coletar as células por centrifugação novamente. </li><li> Ressuspender o sedimento em 500 mL de imediato de água deionizada NanoPure ou buffer. (A suspensão bacteriana deve ser visivelmente turva, a fim de ter uma concentração adequada de células para AFM). </li><li> Aplicar a suspensão de células (10-20 mL) para a mica revestidas de gelatina e espalhe sobre a superfície usando uma ponteira de pipeta (Fig. 1c). Cuidados devem ser tomados para não tocar fisicamente a superfície da gelatina com a ponta da pipeta enquanto aplica ou espalhar a solução bacteriana. </li><li> Permitir que a superfície em repouso por 10 minutos e enxágüe com um fluxo de água ou tampão (Fig. cd 1). Durante todo o procedimento, tome cuidado para assegurar que a amostra não é permitida para secar. </li></ol> 5. Resultados representativos: A Figura 2 mostra imagens de exemplo de células bacterianas que foram montadas em gelatina-revestido mica e fotografada pela AFM. Em nossos laboratórios, amostras são tipicamente fotografada usando um microscópio de força atômica PicoPlus (Agilent Technologies, Temple, AZ) equipado com uma cabeça de digitalização 100 mm. O instrumento é operado em 128-512 pixels por linha de varredura a uma velocidade de varredura de linhas de 0,5-1,0 / s. As vigas que são normalmente utilizados são sondas Veeco nitreto de silício (MLCT-AUHW, Veeco, Santa Barbara, CA) com constantes primavera nominal variando ,01-0,1 nN / nm. <img src="/files/ftp_upload/2880/2880fig1.jpg" alt="Figura 1"/> Figura 1. Gelatina a uma temperatura de 60-70 ° C é colocado em um copo de 20 ml. Usando um par de pinças recém clivada quadrados de mica são totalmente imerso na gelatina (a) retirada e colocada na vertical sobre uma toalha de papel encostado a um rack de micro centrífuga (b). Uma gota de amostra bacteriana é colocada sobre a mica e espalhar com a ponta da pipeta em ambas as direções X e Y (c). Depois de permitir que a amostra para incubar na superfície mica de pelo menos 10 minutos, lavar a amostra em um fluxo de água destilada. Se a imobilização trabalhou você será capaz de ver uma mancha opaca na mica, como mostrado no painel direito de (d). Se a imobilização não trabalho que você vai pegar um pedaço de mica clara como no painel esquerdo. A mancha opaca na balança mica para a concentração das bactérias na gota que você coloque na mica gelatina tratada. <img src="/files/ftp_upload/2880/2880fig2.jpg" alt="Figura 2"/> Imagens figura 2. AFM da E. coli. No painel (a), as células são montadas em gelatina-mica revestidas e fotografada em 0,005 M PBS. Para efeito de comparação, uma imagem de células bacterianas montados em gelatina-mica e revestido com imagens no ar é mostrado no painel (b). Embora septos são claramente visíveis em imagens coletadas no estado líquido, maior a resolução, como indicado pela presença de pili (painel inset (b)), podem ser obtidas no ar.

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+properties+of+axons.">Mechanical properties of axons.</a> Phys Rev Lett. 99, 018301-018301 (2007).</li><li>Bray, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+growth+in+response+to+experimentally+applied+mechanical+tension.">Axonal growth in response to experimentally applied mechanical tension.</a> Dev Biol. 102, 379-389 (1984).</li><li>Chetta, J., Kye, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytoskeletal+dynamics+in+response+to+tensile+loading+of+mammalian+axons.">Cytoskeletal dynamics in response to tensile loading of mammalian axons.</a> Cytoskeleton (Hoboken). 67, 650-665 (2010).</li><li>Dennerll, T. 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Y., Gordon, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cellular+and+molecular+basis+of+peripheral+nerve+regeneration.">The cellular and molecular basis of peripheral nerve regeneration.</a> Mol Neurobiol. 14, 1-2 (1997).</li><li>Gray, C., Hukkanen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+neural+growth:+calcitonin+gene-related+peptide+and+substance+P-+containing+nerves+attain+exceptional+growth+rates+in+regenerating+deer+antler.">Rapid neural growth: calcitonin gene-related peptide and substance P- containing nerves attain exceptional growth rates in regenerating deer antler.</a> Neuroscience. 50, 953-963 (1992).</li><li>Heidemann, S. R., Buxbaum, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tension+as+a+regulator+and+integrator+of+axonal+growth.">Tension as a regulator and integrator of axonal growth.</a> Cell Motil Cytoskeleton. 17, 6-10 (1990).</li><li>Heidemann, S. R., Buxbaum, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+tension+as+a+regulator+of+axonal+development.">Mechanical tension as a regulator of axonal development.</a> Neurotoxicology. 15, 95-107 (1994).</li><li>Heidemann, S. R., Lamoureux, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytomechanics+of+axonal+development.">Cytomechanics of axonal development.</a> Cell Biochem Biophys. 27, 135-155 (1995).</li><li>Iwata, A., Browne, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+survival+and+outgrowth+of+mechanically+engineered+nervous+tissue+constructs+implanted+into+spinal+cord+lesions.">Long-term survival and outgrowth of mechanically engineered nervous tissue constructs implanted into spinal cord lesions.</a> Tissue Eng. 12, 101-110 (2006).</li><li>Lamoureux, P., Heidemann, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+and+elongation+within+and+along+the+axon.">Growth and elongation within and along the axon.</a> Dev Neurobiol. 70, 135-149 (2010).</li><li>Lamoureux, P., Zheng, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cytomechanical+investigation+of+neurite+growth+on+different+culture+surfaces.">A cytomechanical investigation of neurite growth on different culture surfaces.</a> J Cell Biol. 118, 655-661 (1992).</li><li>Lindqvist, N., Liu, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+glial+(Muller)+cells:+sensing+and+responding+to+tissue+stretch.">Retinal glial (Muller) cells: sensing and responding to tissue stretch.</a> Invest Ophthalmol Vis Sci. 51, 1683-1690 (2010).</li><li>Loverde, J. R., Ozoka, V. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+Imaging+of+Axon+Stretch+Growth+in+Embryonic+and+Adult+Neurons.">Live Imaging of Axon Stretch Growth in Embryonic and Adult Neurons.</a> J. Neurotrauma. , (2011).</li><li>Lu, Y. B., Franze, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viscoelastic+properties+of+individual+glial+cells+and+neurons+in+the+CNS.">Viscoelastic properties of individual glial cells and neurons in the CNS.</a> Proc Natl Acad Sci U S A. 103, 17759-17764 (2006).</li><li>O'Toole, M., Lamoureux, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+physical+model+of+axonal+elongation:+force,+viscosity,+and+adhesions+govern+the+mode+of+outgrowth.">A physical model of axonal elongation: force, viscosity, and adhesions govern the mode of outgrowth.</a> Biophys J. 94, 2610-2620 (2008).</li><li>Pfister, B. J., Bonislawski, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stretch-grown+axons+retain+the+ability+to+transmit+active+electrical+signals.">Stretch-grown axons retain the ability to transmit active electrical signals.</a> FEBS Lett. 580, 3525-3531 (2006).</li><li>Pfister, B. J., Gordon, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomedical+Engineering+Strategies+for+Peripheral+Nerve+Repair:+Surgical+Applications,+State+of+the+Art,+and+Future+Challenges.">Biomedical Engineering Strategies for Peripheral Nerve Repair: Surgical Applications, State of the Art, and Future Challenges.</a> Crit Rev Biomed Eng. 39, 81-124 (2011).</li><li>Pfister, B. J., Iwata, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extreme+stretch+growth+of+integrated+axons.">Extreme stretch growth of integrated axons.</a> J Neurosci. 24, 7978-7983 (2004).</li><li>Pfister, B. J., Iwata, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+transplantable+nervous+tissue+constructs+comprised+of+stretch-grown+axons.">Development of transplantable nervous tissue constructs comprised of stretch-grown axons.</a> J Neurosci Methods. 153, 95-103 (2006).</li><li>Siechen, S., Yang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+tension+contributes+to+clustering+of+neurotransmitter+vesicles+at+presynaptic+terminals.">Mechanical tension contributes to clustering of neurotransmitter vesicles at presynaptic terminals.</a> Proc Natl Acad Sci U S A. 106, 12611-12616 (2009).</li><li>Smith, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stretch+growth+of+integrated+axon+tracts:+extremes+and+exploitations.">Stretch growth of integrated axon tracts: extremes and exploitations.</a> Prog Neurobiol. 89, 231-239 (2009).</li><li>Smith, D. H., Wolf, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+strategy+to+produce+sustained+growth+of+central+nervous+system+axons:+continuous+mechanical+tension.">A new strategy to produce sustained growth of central nervous system axons: continuous mechanical tension.</a> Tissue Eng. 7, 131-139 (2001).</li><li>Weiss, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nerve+patterns:+The+mechanics+of+nerve+growth.">Nerve patterns: The mechanics of nerve growth.</a> Growth, Third Growth Symposium. 5, 163-203 (1941).</li><li>Zheng, J., Lamoureux, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tensile+regulation+of+axonal+elongation+and+initiation.">Tensile regulation of axonal elongation and initiation.</a> J Neurosci. 11, 1117-1125 (1991).</li></ol>

Não há conflitos de interesse declarados.

Vários fatores podem afetar célula microbiana de montagem e de imagem por AFM. A gelatina que é usado para o revestimento da mica é importante. Comercial de gelatina é isolado a partir de um número de vertebrados, incluindo peixes, vacas e porcos. Tanto a origem eo método de processamento de determinar a adequação da gelatina para imobilização de bactérias. Várias fontes e tipos de gelatina foram avaliados quanto à sua eficácia em bactérias imobilizar [1]. As duas gelatinas mais eficazes foram encontrado...

<table border="1"><tbody><tr><td> Nome </td><td> Companhia </td><td> Número de catálogo </td></tr><tr><td> Gelatina </td><td> Sigma, St. Louis, MO </td><td> G6144, G2625 ou G2500 </td></tr><tr><td> PicoPlus Microscópio de Força Atômica </td><td> Agilent Technologies, Tempe, AZ </td><td></td></tr><tr><td> Cantilevers AFM </td><td> Veeco, Santa Barbara, CA </td><td> MLCT-AUHW </td></tr></tbody></table>

bacterial immobilization for imaging by atomic force microscopy

AFM é um de alta resolução (escala nm) ferramenta de imagem que mecanicamente um sondas de superfície. Ele tem a capacidade de células e biomoléculas imagem, em um ambiente líquido, sem a necessidade de tratar quimicamente a amostra. A fim de atingir esse objetivo, a amostra deve suficientemente aderir à superfície de montagem para evitar a remoção por forças exercidas pela ponta cantilever digitalização AFM. Em muitos casos, a imagem de sucesso depende de imobilização da amostra à superfície de montagem. Idealmente, a imobilização deve ser minimamente invasivo para a amostra de tal forma que os processos metabólicos e atributos funcionais não sejam comprometidas. Por superfícies de revestimento recém clivada com mica suína (porco) gelatina, bactérias carga negativa pode ser imobilizada na superfície e fotografada em líquido por AFM. Imobilização de células bacterianas em gelatina-mica revestida é mais provável devido à interação eletrostática entre as bactérias com carga negativa e positivamente carregada a gelatina. Vários fatores podem interferir com a imobilização de bactérias, incluindo componentes químicos do líquido em que as bactérias estão suspensos, o tempo de incubação da bactéria na gelatina mica revestidas características, superfície da estirpe bacteriana e do meio em que as bactérias são gravadas. No geral, o uso de gelatina revestido mica é encontrado para ser geralmente aplicável para as células microbianas imagem.

Watch this Scientific Journal Video about Bacterial Immobilization for Imaging by Atomic Force Microscopy at JoVE.com

Bacterial Immobilization for Imaging by Atomic Force Microscopy

Vivem as bactérias Gram-negativas e Gram-positivos podem ser imobilizadas em gelatina-mica revestidas com imagens e em líquido usando Microscopia de Força Atômica (AFM).

A imobilização de bactérias for Imaging por Microscopia de Força Atômica

AFM is a high-resolution (nm scale) imaging tool that mechanically probes a surface.  It has the ability to image cells and biomolecules, in a liquid ...

bacterial-immobilization-for-imaging-by-atomic-force-microscopy

Research

JoVE Journal

Bioengineering

17.3K visualizações.  Oak Ridge National Laboratory. Vivem as bactérias Gram-negativas e Gram-positivos podem ser imobilizadas em gelatina-mica revestidas com imagens e em líquido usando Microscopia de Força Atômica (AFM).

Vídeo: Bacterial Immobilization for Imaging by Atomic Force Microscopy

Three-dimensional Optical-resolution Photoacoustic Microscopy

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As the mainstays of in vivo three-dimensional (3-D) optical microscopy, single-/multi-photon fluorescence microscopy and optical coherence tomography (OCT) have demonstrated their extraordinary sensitivities to fluorescence and optical scattering contrasts, respectively. However, the optical absorption contrast of biological tissues, which encodes essential physiological/pathological information, has not yet been assessable. 
The emergence of biomedical photoacoustics has led to a new branch of optical microscopy optical-resolution photoacoustic microscopy (OR-PAM)1, where the optical irradiation is focused to the diffraction limit to achieve cellular1 or even subcellular2 level lateral resolution. As a valuable complement to existing optical microscopy technologies, OR-PAM brings in at least two novelties. First and most importantly, OR-PAM detects optical absorption contrasts with extraordinary sensitivity (i.e., 100%). Combining OR-PAM with fluorescence microscopy3 or with optical-scattering-based OCT4 (or with both) provides comprehensive optical properties of biological tissues. Second, OR-PAM encodes optical absorption into acoustic waves, in contrast to the pure optical processes in fluorescence microscopy and OCT, and provides background-free detection. The acoustic detection in OR-PAM mitigates the impacts of optical scattering on signal degradation and naturally eliminates possible interferences (i.e., crosstalks) between excitation and detection, which is a common problem in fluorescence microscopy due to the overlap between the excitation and fluorescence spectra. 
Unique for optical absorption imaging, OR-PAM has demonstrated broad biomedical applications since its invention, including, but not limited to, neurology5, 6, ophthalmology7, 8, vascular biology9, and dermatology10. In this video, we teach the system configuration and alignment of OR-PAM as well as the experimental procedures for in vivo functional microvascular imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology capable of imaging optical absorption contrasts in vivo with cellular resolution and sensitivity. Here, we provide a visualized instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, typical in vivo experimental procedures, and functional imaging schemes.

Tridimensional Optical resolução Microscopia Fotoacústica

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As ...

Bioengenharia

Visualization of Recombinant DNA and Protein Complexes Using Atomic Force Microscopy

Atomic force microscopy (AFM) allows for the visualizing of individual proteins, DNA molecules, protein-protein complexes, and DNA-protein complexes. On the end of the microscope's cantilever is a nano-scale probe, which traverses image areas ranging from nanometers to micrometers, measuring the elevation of macromolecules resting on the substrate surface at any given point. Electrostatic forces cause proteins, lipids, and nucleic acids to loosely attach to the substrate in random orientations and permit imaging. The generated data resemble a topographical map, where the macromolecules resolve as three-dimensional particles of discrete sizes (Figure 1) 1,2. Tapping mode AFM involves the repeated oscillation of the cantilever, which permits imaging of relatively soft biomaterials such as DNA and proteins. One of the notable benefits of AFM over other nanoscale microscopy techniques is its relative adaptability to visualize individual proteins and macromolecular complexes in aqueous buffers, including near-physiologic buffered conditions, in real-time, and without staining or coating the sample to be imaged. 
The method presented here describes the imaging of DNA and an immunoadsorbed transcription factor (i.e. the glucocorticoid receptor, GR) in buffered solution (Figure 2). Immunoadsorbed proteins and protein complexes can be separated from the immunoadsorbing antibody-bead pellet by competition with the antibody epitope and then imaged (Figure 2A). This allows for biochemical manipulation of the biomolecules of interest prior to imaging. Once purified, DNA and proteins can be mixed and the resultant interacting complex can be imaged as well. Binding of DNA to mica requires a divalent cation 3,such as Ni2+ or Mg2+, which can be added to sample buffers yet maintain protein activity. Using a similar approach, AFM has been utilized to visualize individual enzymes, including RNA polymerase 4 and a repair enzyme 5, bound to individual DNA strands. These experiments provide significant insight into the protein-protein and DNA-protein biophysical interactions taking place at the molecular level. Imaging individual macromolecular particles with AFM can be useful for determining particle homogeneity and for identifying the physical arrangement of constituent components of the imaged particles. While the present method was developed for visualization of GR-chaperone protein complexes 1,2 and DNA strands to which the GR can bind, it can be applied broadly to imaging DNA and protein samples from a variety of sources.

A tapping mode atomic force microscope (AFM) method for the visualization of plasmid DNA, cytoplasmic proteins, and DNA-protein complexes is described. The method includes alternate approaches for preparing samples for AFM imaging following biochemical manipulation. DNA containing specific protein interacting regions are observed in near-physiologic buffer conditions.

Visualização do DNA recombinante e Complexos de Proteínas Usando Microscopia de Força Atômica

Atomic force microscopy (AFM) allows for the visualizing of individual proteins, DNA molecules, protein-protein complexes, and DNA-protein complexes. On ...

Quantifying the Mechanical Properties of the Endothelial Glycocalyx with Atomic Force Microscopy

Our understanding of the interaction of leukocytes and the vessel wall during leukocyte capture is limited by an incomplete understanding of the mechanical properties of the endothelial surface layer. It is known that adhesion molecules on leukocytes are distributed non-uniformly relative to surface topography 3, that topography limits adhesive bond formation with other surfaces 9, and that physiological contact forces (&asymp; 5.0 &minus; 10.0 pN per microvillus) can compress the microvilli to as little as a third of their resting length, increasing the accessibility of molecules to the opposing surface 3, 7. We consider the endothelium as a two-layered structure, the relatively rigid cell body, plus the glycocalyx, a soft protective sugar coating on the luminal surface 6. It has been shown that the glycocalyx can act as a barrier to reduce adhesion of leukocytes to the endothelial surface 4. In this report we begin to address the deformability of endothelial surfaces to understand how the endothelial mechanical stiffness might affect bond formation. Endothelial cells grown in static culture do not express a robust glycocalyx, but cells grown under physiological flow conditions begin to approximate the glycocalyx observed in vivo 2. The modulus of the endothelial cell body has been measured using atomic force microscopy (AFM) to be approximately 5 to 20 kPa 5. The thickness and structure of the glycocalyx have been studied using electron microscopy 8, and the modulus of the glycocalyx has been approximated using indirect methods, but to our knowledge, there have been no published reports of a direct measurement of the glycocalyx modulus in living cells. In this study, we present indentation experiments made with a novel AFM probe on cells that have been cultured in conditions to maximize their glycocalyx expression to make direct measurements of the modulus and thickness of the endothelial glycocalyx.

The mechanical characteristics of endothelial glycocalyx were measured by indentation using micron sized spheres on AFM cantilevers. Endothelial cells were cultured in a custom chamber under physiological flow conditions to induce glycocalyx expression. Data were analyzed using a thin film model to determine the glycocalyx thickness and modulus.

Quantificar as propriedades mecânicas do Glicocálix endotelial com Microscopia de Força Atômica

Our  understanding of the interaction of leukocytes and the vessel wall during  leukocyte capture is limited by an incomplete understanding of the ...

Measuring the Mechanical Properties of Living Cells Using Atomic Force Microscopy

Mechanical properties of cells and extracellular matrix (ECM) play important roles in many biological processes including stem cell differentiation, tumor formation, and wound healing. Changes in stiffness of cells and ECM are often signs of changes in cell physiology or diseases in tissues. Hence, cell stiffness is an index to evaluate the status of cell cultures. Among the multitude of methods applied to measure the stiffness of cells and tissues, micro-indentation using an Atomic Force Microscope (AFM) provides a way to reliably measure the stiffness of living cells. This method has been widely applied to characterize the micro-scale stiffness for a variety of materials ranging from metal surfaces to soft biological tissues and cells. The basic principle of this method is to indent a cell with an AFM tip of selected geometry and measure the applied force from the bending of the AFM cantilever. Fitting the force-indentation curve to the Hertz model for the corresponding tip geometry can give quantitative measurements of material stiffness. This paper demonstrates the procedure to characterize the stiffness of living cells using AFM. Key steps including the process of AFM calibration, force-curve acquisition, and data analysis using a MATLAB routine are demonstrated. Limitations of this method are also discussed.

This paper demonstrates a protocol to characterize the mechanical properties of living cells by means of microindentation using an Atomic Force Microscope (AFM).

Medir as propriedades mecânicas das células vivas Usando Microscopia de Força Atômica

Mechanical  properties of cells and extracellular matrix (ECM) play important roles in many  biological processes including stem cell differentiation, ...

High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., kinetics, coexistence of different states and populations, transient interactions), which are typically hidden in ensemble measurements, such as those obtained with standard biochemical or microscopy methods. Thus, dynamic events, such as receptor-receptor interactions, can be followed in real time in a living cell with high spatiotemporal resolution. This protocol describes a method based on labeling with small and bright organic fluorophores and total internal reflection fluorescence (TIRF) microscopy to directly visualize single receptors on the surface of living cells. This approach allows one to precisely localize receptors, measure the size of receptor complexes, and capture dynamic events such as transient receptor-receptor interactions. The protocol provides a detailed description of how to perform a single-molecule experiment, including sample preparation, image acquisition and image analysis. As an example, the application of this method to analyze two G-protein-coupled receptors, i.e., &#946;2-adrenergic and &#947;-aminobutyric acid type B (GABAB) receptor, is reported. The protocol can be adapted to other membrane proteins and different cell models, transfection methods and labeling strategies.

This protocol describes how to use total internal reflection fluorescence microscopy to visualize and track single receptors on the surface of living cells and thereby analyze receptor lateral mobility, size of receptor complexes as well as to visualize transient receptor-receptor interactions. This protocol can be extended to other membrane proteins.

De alta resolução espacial e temporal Análise de Receptor Dynamics por uma única molécula de microscopia de fluorescência

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., ...

Novel Atomic Force Microscopy Based Biopanning for Isolation of Morphology Specific Reagents against TDP-43 Variants in Amyotrophic Lateral Sclerosis

Because protein variants play critical roles in many diseases including TDP-43 in Amyotrophic Lateral Sclerosis (ALS), alpha-synuclein in Parkinson&#8217;s disease and beta-amyloid and tau in Alzheimer&#8217;s disease, it is critically important to develop morphology specific reagents that can selectively target these disease-specific protein variants to study the role of these variants in disease pathology and for potential diagnostic and therapeutic applications. We have developed novel atomic force microscopy (AFM) based biopanning techniques that enable isolation of reagents that selectively recognize disease-specific protein variants. There are two key phases involved in the process, the negative and positive panning phases. During the negative panning phase, phages that are reactive to off-target antigens are eliminated through multiple rounds of subtractive panning utilizing a series of carefully selected off-target antigens. A key feature in the negative panning phase is utilizing AFM imaging to monitor the process and confirm that all undesired phage particles are removed. For the positive panning phase, the target antigen of interest is fixed on a mica surface and bound phages are eluted and screened to identify phages that selectively bind the target antigen. The target protein variant does not need to be purified providing the appropriate negative panning controls have been used. Even target protein variants that are only present at very low concentrations in complex biological material can be utilized in the positive panning step. Through application of this technology, we acquired antibodies to protein variants of TDP-43 that are selectively found in human ALS brain tissue. We expect that this protocol should be applicable to generating reagents that selectively bind protein variants present in a wide variety of different biological processes and diseases.

Using atomic force microscopy in combination with biopanning technology we created a negative and positive biopanning system to acquire antibodies against disease-specific protein variants present in any biological material, even at low concentrations. We were successful in obtaining antibodies to TDP-43 protein variants involved in Amyotrophic Lateral Sclerosis.

Novel Microscopia de Força Atômica bioprospecção Based para Isolamento de Morfologia reagentes específicos contra TDP-43 variantes em Esclerose Lateral Amiotrófica

Because protein variants play critical roles in many diseases including TDP-43 in Amyotrophic Lateral Sclerosis (ALS), alpha-synuclein in ...

Atomic Force Microscopy Imaging and Force Spectroscopy of Supported Lipid Bilayers

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient magnification to examine membrane substructures and even individual molecules. AFM can act as a force probe to measure interactions and mechanical properties of membranes. Supported lipid bilayers are conventionally used as membrane models in AFM studies. In this protocol, we demonstrate how to prepare supported bilayers and characterize their structure and mechanical properties using AFM. These include bilayer thickness and breakthrough force. 
The information provided by AFM imaging and force spectroscopy help define mechanical and chemical properties of membranes. These properties play an important role in cellular processes such as maintaining cell hemostasis from environmental stress, bringing membrane proteins together, and stabilizing protein complexes.

We describe a protocol for preparation of supported lipid bilayers and its characterization using atomic force microscopy and force spectroscopy.

Projeção de imagem atômica e força Espectroscopia de Bicamadas Lipídicas suportados

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient ...

Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair

The reconstruction of critically sized bone defects remains a serious clinical problem because of poor angiogenesis within tissue-engineered scaffolds during repair, which gives rise to a lack of sufficient blood supply and causes necrosis of the new tissues. Rapid vascularization is a vital prerequisite for new tissue survival and integration with existing host tissue. The de novo generation of vasculature in scaffolds is one of the most important steps in making bone regeneration more efficient, allowing repairing tissue to grow into a scaffold. To tackle this problem, the genetic modification of a biomaterial scaffold is used to accelerate angiogenesis and osteogenesis. However, visualizing and tracking in vivo blood vessel formation in real-time and in three-dimensional (3D) scaffolds or new bone tissue is still an obstacle for bone tissue engineering. Multiphoton microscopy (MPM) is a novel bio-imaging modality that can acquire volumetric data from biological structures in a high-resolution and minimally-invasive manner. The objective of this study was to visualize angiogenesis with multiphoton microscopy in vivo in a genetically modified 3D-PLGA/nHAp scaffold for calvarial critical bone defect repair. PLGA/nHAp scaffolds were functionalized for the sustained delivery of a growth factor pdgf-b gene carrying lentiviral vectors (LV-pdgfb) in order to facilitate angiogenesis and to enhance bone regeneration. In a scaffold-implanted calvarial critical bone defect mouse model, the blood vessel areas (BVAs) in PHp scaffolds were significantly higher than in PH scaffolds. Additionally, the expression of pdgf-b and angiogenesis-related genes, vWF and VEGFR2, increased correspondingly. MicroCT analysis indicated that the new bone formation in the PHp group dramatically improved compared to the other groups. To our knowledge, this is the first time multiphoton microscopy was used in bone tissue-engineering to investigate angiogenesis in a 3D bio-degradable scaffold in vivo and in real-time.

Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair

Here, we present a protocol to visualize blood vessel formation in vivo and in real-time in 3D scaffolds by multiphoton microscopy. Angiogenesis in genetically modified scaffolds was studied in a murine calvarial critical bone defect model. More new blood vessels were detected in the treatment group than in controls.

Visualizando a angiogênese por Multiphoton microscopia In Vivo no andaime de 3D-PLGA/nhập geneticamente modificado para raspagem crítico reparo de defeito ósseo

The reconstruction of critically sized bone defects remains a serious clinical problem because of poor angiogenesis within tissue-engineered scaffolds ...

Visualizing Adhesion Formation in Cells by Means of Advanced Spinning Disk-Total Internal Reflection Fluorescence Microscopy

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to visualize these highly dynamic events, two complementary light microscopy techniques that allow fast imaging of live samples were combined: spinning disk microscopy (SD) for fast and high-resolution volume recording and total internal reflection fluorescence (TIRF) microscopy for precise localization and visualization of the plasma membrane. A comprehensive and complete imaging protocol will be shown for guiding through sample preparation, microscope calibration, image formation and acquisition, resulting in multi-color SD-TIRF live imaging series with high spatio-temporal resolution. All necessary image post-processing steps to generate multi-dimensional live imaging datasets, i.e. registration and combination of the individual channels, are provided in a self-written macro for the open source software ImageJ. The imaging of fluorescent proteins during initiation and maturation of adhesion complexes, as well as the formation of the actin cytoskeletal network, was used as a proof of principle for this novel approach. The combination of high resolution 3D microscopy and TIRF provided a detailed description of these complex processes within the cellular environment and, at the same time, precise localization of the membrane-associated molecules detected with a high signal-to-background ratio.

An advanced microscope that permit fast and high-resolution imaging of both, the isolated plasma membrane and the surrounding intracellular volume, will be presented. The integration of spinning disk and total internal reflection fluorescence microscopy in one setup allows live imaging experiments at high acquisition rates up to 3.5 s per image stack.

Visualizando a formação de adesão em células por meio de avançados girando a reflexão interna Total disco microscopia de fluorescência

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to ...

Application of Atomic Force Microscopy to Detect Early Osteoarthritis

Biomechanical properties of cells and tissues not only regulate their shape and function but are also crucial for maintaining their vitality. Changes in elasticity can propagate or trigger the onset of major diseases like cancer or osteoarthritis (OA). Atomic force microscopy (AFM) has emerged as a strong tool to qualitatively and quantitatively characterize the biomechanical properties of specific biological target structures on a microscopic scale, measuring forces in a range from as small as the piconewton to the micronewton. Biomechanical properties are of special importance in musculoskeletal tissues, which are subjected to high levels of strain. OA as a degenerative disease of the cartilage results in the disruption of the pericellular matrix (PCM) and the spatial rearrangement of the chondrocytes embedded in their extracellular matrix (ECM). Disruption in PCM and ECM has been associated with changes in the biomechanical properties of cartilage. In the present study we used AFM to quantify these changes in relation to the specific spatial pattern changes of the chondrocytes. With each pattern change, significant changes in elasticity were observed for both the PCM and ECM. Measuring the local elasticity thus allows for drawing direct conclusions about the degree of local tissue degeneration in OA.

We present a method to investigate early osteoarthritic changes at the cellular level in articular cartilage by using atomic force microscopy (AFM).

Aplicação de microscopia de força atômica para detectar osteoartrite precoce

Biomechanical properties of cells and tissues not only regulate their shape and function but are also crucial for maintaining their vitality. Changes in ...